Effects of locally formed angiotensin II on renal hemodynamics

The kidney produces angiotensin II (AngII) by conversion of both locally formed and systemically delivered angiotensin I (AngI). The latter may be physiologically significant because the kidney can convert 20-25% of systemically delivered AngI. To determine possible differences between the effects of circulating and locally converted AngII, we compared the renal responses to renal arterial infusions of AngI and AngII in equiconstrictor doses. Both reduced the renal blood flow and increased the filtration fraction; it is important that the AngI infusions consistently reduced glomerular filtration rates (GFR), which indicates effects proximal to or at the glomerulus. Micropuncture experiments revealed that AngI infusions reduced proximal tubular and peritubular capillary pressures and the single-nephron GFR; glomerular capillary pressure was not altered significantly. AngI infusions increased both pre- and postglomerular resistances and reduced the glomerular filtration coefficient. In other studies designed to estimate net intrarenal AngII generation, it was determined that the kidney degrades about 90% of arterially delivered AngII. Thus, most of the AngII in renal venous blood was formed intrarenally. Local production of AngII was enhanced, in association with increased renin release, after reductions in renal arterial pressure. Such increases in intrarenal AngII production may contribute to the AngII-dependent changes in renal vascular resistance that occur in conditions where the renin-angiotensin system is stimulated.     

Renin substrate in granules from rat kidney cortex.

1. Subcellular fractions of rat kidney cortex generated angiotensin I continuously over 2h when incubated at 37degreesC with rat renin, indicating the presence of renin substrate within cells in the renal cortex. 2. Renin substrate was located in highest specific concentration in particulate fractions. The particles containing renin substrate had a sedimentation velocity slightly lower than mitochondria and renin granules but greater than the microsomal fraction. 3. Isopycnic gradient centrifugation indicated a density of 1.190g/ml for the particles containing renin substrate, compared with 1.201 for renin granules, 1.177 for mitochondria, and 1.170 and 1.230 for lysosomes in the heavy-granule fraction. 4. In the liver, renin substrate was also found in particles, but these had a lower sedimentation rate than those from the kidney. 5. The molecular weights of renin substrate in kidney and liver granules and rat plasma were similar, namely 61000-62000. 6. On the basis of these biochemical findings, a mechanism for the intrarenal production of angiotensin, incorporating a subcellular reaction scheme, is proposed.     

Differential ACE expression among tissues in allele-specific Wistar rat lines

In humans, the insertion/deletion polymorphism in the angiotensin converting enzyme (ACE) gene accounts for half of the variance in plasma ACE activity. The deletion allele is associated with high plasma ACE activity, cardiovascular disease, and renal disease. In rat, a similar association is found between the B and L alleles of a microsatellite marker in the ACE gene. We identified the B/L variation in the Wistar outbred rat and bred two lines homozygous for the two alleles (WU-B and WU-L). ACE activity was measured in serum, heart, kidney, and aorta homogenates. Immunohistochemistry and ACE mRNA expression were performed in heart, kidney, and aortic tissue. Aortic rings were collected and stimulated with AngI, AngII, and AngI with Lisinopril to measure ACE functional activity by vasoconstrictor response. Serum, heart, and kidney ACE activity and kidney mRNA expression were two-fold higher in WU-B. Kidney staining showed a clear difference in tubular ACE expression, with more staining in WU-B. While in aorta ACE activity and mRNA expression was twofold higher in WU-L, functional conversion of AngI was higher in WU-B, indicating either a functional difference in AngI to AngII conversion between the two alleles due to different splicing or the presence of other factors involved in the conversion that are differentially expressed as the result of differences in the ACE alleles. The newly developed WU-B and WU-L lines show tissue-specific differences in ACE expression and activity. This provides an experimental tool to study the pathophysiologic consequences of differences in ACE alleles in renal and cardiovascular disease. J. Kamilic and A. T. Lely contributed equally to this work.     

Duplexed iMALDI for the detection of angiotensin I and angiotensin II

An immuno-Matrix Assisted Laser Desorption/Ionization (iMALDI) method has been developed using anti-IgG beads to capture anti-AngI and anti-AngII antibodies, which are incubated with a ～50μL plasma sample to which known amounts of stable-isotope-labeled AngI and AngII have been added. After a short incubation time, the beads are washed, placed directly on a MALDI target, and analyzed by mass spectrometry (MS) and tandem mass spectrometry (MS/MS). The iMALDI assay developed can detect and quantify angiotensin I (AngI) and angiotensin II (AngII) in human plasma. This assay has a Limit of Detection (LOD) of ～10amol/μL (or ～13pg/mL AngI and ～11pg/mL AngII), at a S/N of 2:1, using only one-tenth of the antibody beads which were incubated with a 50-μL plasma sample. This LOD is within the relevant range of patient samples. Little or no angiotensin generation period is required, resulting in a rapid assay. Correlation coefficients for the standard curves are >0.99, with a linear range of 4-100fmol/μL (5-130ng/mL) and 100-2500amol/μL (106-2614pg/mL) for AngI and AngII, respectively. This duplexed assay can quantify AngI and AngII peptide levels simultaneously, in plasma from normotensive and hypertensive patients. The assay can detect changes in the levels of these peptides over time, which will allow quantitation of plasma renin and ACE activities.     

Structure and reorientational dynamics of angiotensin I and II: a microscopic physical insight

We present a study of structural analysis and reorientational dynamics of Angiotensin I (AngI) and Angiotensin II (AngII) in aqueous solution. AngI is a decapeptide that acts as a precursor to the octapeptide AngII in the Renin-Angiotensin-Aldosterone system for blood pressure regulation. Experimental structural characterization of these peptides, carried out with circular dichroism and infrared spectroscopy, showed that the angiotensins are mostly disordered but exhibit a measurable population of ordered structures at room temperature. Interestingly, these change from the unordered polyproline-like conformation for AngI to a more compact and ordered conformation for AngII as the length of the peptide is decreased. Anisotropy decay measurements with picosecond time resolution indicate slower overall tumbling and a greater amplitude of internal motion in AngI compared to AngII, consistent with more compact and less flexible structure of the active form of the peptide. To model the microscopic behavior of the peptides, 2-μs molecular dynamics simulation trajectories were generated for AngI and AngII, at 300?K using the OPLS-AA potential and SPC water. The structures sampled in the simulations mostly agree with the experimental results, showing the prevalence of disordered structures, turns, and polyproline helices. Additionally, the computational results predict fewer sampled conformations, tighter side-chain packing and marked increase of Phe8 solvent accessibility upon AngI truncation to AngII. Our combined approach of experiment and extensive computer simulation thus yields new information on the conformational dynamics of the angiotensins, helping provide insight into the structural basis for the potency of AngI relative to AngII.     

Distinct localization of renin and angiotensins in separate subcellular fractions of the rat adrenal cortex.

The subcellular localization of renin and immunoreactive angiotensins I and II was studied in rat adrenal cortical tissues. The identity of the immunoreactive angiotensins was confirmed as angiotensin I and angiotensin II by radioimmunoassay and high-performance liquid chromatography, respectively, with reference to standard compounds. By differential centrifugation of tissue homogenate in 0.25 M sucrose/30 mM Tris-HCl/l mM EDTA, pH 7.4, specific immunoreactive renin was found to be localized principally (60%) in the mitochondrial fraction (P2), whereas about 40% of both angiotensins I and II was contained in the soluble fraction; only 18-20% of both peptides was contained in the P2 fraction. On Percoll density gradient centrifugation of P2, renin was fractionated mostly in a denser band whereas angiotensins I and II were contained in a lighter density area closely corresponding to mitochondrial and lysosomal marker enzymes. These results suggest that renin and angiotensins in the cells of the rat adrenal gland reside in different subcellular compartments and argue against intracellular formation of angiotensins by renin in renin granules.     

Comparative effects of angiotensin IV and two hemorphins on angiotensin-converting enzyme activity

The role of angiotensin IV (AngIV) in the regulation of angiotensin-converting enzyme (ACE) was studied in vitro. This study demonstrates that this active fragment appeared as a novel endogenous ACE inhibitor. Inhibitory kinetic studies revealed that AngIV acts as a purely competitive inhibitor with a K(i) value of 35 microM. AngIV was found to be quite resistant to ACE hydrolysis opposite to hemorphins which are both ACE inhibitors and substrates. In order to confirm a putative role of AngIV and hemorphins in the Renin-Angiotensin system (RAS) regulation, we studied their influence on AngI conversion. We noticed that 16.7 microM of both peptides decreased more than 50% of AngI conversion to AngII in vitro. The capacity of hemorphins, particularly LVVH-7, and AngIV to inhibit ACE activity here suggests a synergistic relation between these two peptides and the regulation of RAS.     

Functional role of local angiotensin-converting enzyme (ACE) in adrenal catecholamine secretion in vivo

The aim of the present study was to investigate whether exogenous angiotensin I (AngI) is locally converted to angiotensin II (AngII), which in turn results in an increase in the adrenal catecholamine (CA) secretion in the adrenal gland in anesthetized dogs. Plasma CA concentrations in adrenal venous and aortic blood were determined by an HPLC-electrochemical method. Adrenal venous blood flow was measured by gravimetry. Local administration of AngI (0.0062 to 6.2 microg, 0.0096 to 9.6 microM) to the left adrenal gland resulted in significant increases in CA output in a dose-dependent manner. Following administration of 0.62 microg (0.96 microM) of AngI, adrenal epinephrine and norepinephrine outputs increased from 20.8+/-13.6 to 250.9+/-96.4 ng x min(-1) x g(-1) (p<0.05, n = 5) and from 2.8+/-1.7 to 29.6+/-11.1 ng x min(-1) x g(-1) (p<0.05, n = 5), respectively. From the same left adrenal gland, the output of AngII increased from -0.02+/-0.04 to 26.39+/-11.38 ng x min(-1) x g(-1) (p<0.05, n = 5), while plasma concentrations of AngII in aortic blood remained unchanged. In dogs receiving captopril (12.5 microg, 0.5 mM) 10 min prior to AngI, the net amounts of CA and AngII secreted during the first 3 min after AngI were diminished by about 80% (p<0.05, n = 5) compared with those obtained from the control group. There was a close correlation (r2 = 0.91, n = 6) between the net increases in AngII and CA outputs induced by AngI. The results indicate that the local angiotensin converting enzyme is functionally involved in regional AngII formation in the canine adrenal gland in vivo. The study suggests that AngII thus generated may play a role in the local regulation of adrenal CA secretion.     

The levels of renin-angiotensin related components are modified in the hippocampus of rats submitted to pilocarpine model of epilepsy

We previously showed that patients with temporal lobe epilepsy (TLE) present an increased expression of angiotensin II (AngII) AT1 and AT2 receptors in the hippocampus, supporting the idea of an upregulation of renin-angiotensin system (RAS) in this disease. This study aimed to verify the relationship between the RAS and TLE during epileptogenesis. Levels of the peptides angiotensin I (AngI), angiotensin II (AngII) and angiotensin 1-7 (Ang 1-7), were detected by HPLC assay. Angiotensin AT1 and AT2 receptors, Mas mRNA receptors and angiotensin converting enzyme (ACE), tonin and neutral endopeptidase (NEP) mRNA were also quantified at the hippocampus of Wistar rats by real time PCR, during acute (n=10), silent (n=10) and chronic (n=10) phases of pilocarpine-induced epilepsy. We observed an increased peptide level of Ang1-7 into acute and silent phases, decreasing importantly (p≤0.05) in the chronic phase, suggesting that AngI may be converted into Ang 1-7 by NEP, which is present in high levels in these periods. Our results also showed increased peptide level of AngII in the chronic phase of this model. In contraposition, the ACE expression is reduced in all periods. These data suggest that angiotensinogen or AngI may be cleaved to AngII by tonin, which showed increased expression in all phases. We found changes in AT1, AT2 and Mas mRNA receptors levels suggesting that Ang1-7 could act at Mas receptor during the silent period. Herein, we demonstrated for the first time, changes in angiotensin-related peptides, their receptors as well as the releasing enzymes in the hippocampus of rats during pilocarpine-induced epilepsy.     

Vesicle-associated membrane protein-2 (VAMP2) mediates cAMP-stimulated renin release in mouse juxtaglomerular cells

Renin is essential for blood pressure control. Renin is stored in granules in juxtaglomerular (JG) cells, located in the pole of the renal afferent arterioles. The second messenger cAMP stimulates renin release. However, it is unclear whether fusion and exocytosis of renin-containing granules is involved. In addition, the role of the fusion proteins, SNAREs (soluble N-ethylmaleimide-sensitive factor attachment proteins), in renin release from JG cells has not been studied. The vesicle SNARE proteins VAMP2 (vesicle associated membrane protein 2) and VAMP3 mediate cAMP-stimulated exocytosis in other endocrine cells. Thus, we hypothesized that VAMP2 and/or -3 mediate cAMP-stimulated renin release from JG cells. By fluorescence-activated cell sorting, we isolated JG cells expressing green fluorescent protein and compared the relative abundance of VAMP2/3 in JG cells versus total mouse kidney mRNA by quantitative PCR. We found that VAMP2 and VAMP3 mRNA are expressed and enriched in JG cells. Confocal imaging of primary cultures of JG cells showed that VAMP2 (but not VAMP3) co-localized with renin-containing granules. Cleavage of VAMP2 and VAMP3 with tetanus toxin blocked cAMP-stimulated renin release from JG cells by ~50% and impaired cAMP-stimulated exocytosis by ~50%, as monitored with FM1-43. Then we specifically knocked down VAMP2 or VAMP3 by adenoviral-mediated delivery of short hairpin silencing RNA. We found that silencing VAMP2 blocked cAMP-induced renin release by ~50%. In contrast, silencing VAMP3 had no effect on basal or cAMP-stimulated renin release. We conclude that VAMP2 and VAMP3 are expressed in JG cells, but only VAMP2 is targeted to renin-containing granules and mediates the stimulatory effect of cAMP on renin exocytosis.     

The Bradykinin B2 receptor is required for full expression of renal COX-2 and renin

Angiotensin converting enzyme (ACE) inhibition leads to increased levels of bradykinin, cyclooxygenase-2 (COX-2), and renin. Since bradykinin stimulates prostaglandin release, renin synthesis may be regulated through a kinin-COX-2 pathway. To test this hypothesis, we examined the impact of bradykinin B2 receptor (B2R) gene disruption in mice on kidney COX-2 and renin gene expression. Kidney COX-2 mRNA and protein levels were significantly lower in B2R-/- mice by 40-50%. On the other hand, renal COX-1 levels were similar in B2R-/- and +/+ mice. Renal renin protein was 61% lower in B2R-/- compared to B2R+/+ mice. This was accompanied by a significant reduction in renin mRNA levels in B2R-/- mice. Likewise, intrarenal angiotensin I levels were significantly lower in B2R-/- mice compared to B2R+/+ mice. In contrast, kidney angiotensin II levels were not different and averaged 261+/-16 and 266+/-15fmol/g in B2R+/+ and B2R-/- mice, respectively. Kidney angiotensinogen, AT1 receptor and ACE activity were not different between B2R+/+ and B2R-/- mice. The results of these studies demonstrate suppression of renal renin synthesis in mice lacking the bradykinin B2R and support the notion that B2R regulation of COX-2 participates in the steady-state control of renin gene expression.     

The Renin-Angiotensin System in Fishes

It has been suggested that the renin-angiotensin system (RAS)in mammals may participate in the control of blood pressure,regulation of aldosterone secretion, or in renal functions byinfluencing intrarenal hemodynamics, or possibly by directlyaltering renal tubular sodium reabsorption. Comparative studieshave shown that this system is present among most vertebrates.Renal renin activity and juxtaglomerular cells (JGC), the possiblesite of formation and accumulation of renin, have not been foundin the cyclostomes and elasmobranchs. They seem to have evolvedin primitive bony fishes, being present in all living groupsof actinopterygians and sarcopterygians. Both renin and JGCmay also exist in a holocephalian, the ratfish, Hydrolagus colliei.The functions of the RAS are not yet denned in fishes. Thereis no clear evidence for sodium retaining function of the RASin fishes. Fish angiotensins (angiotensin-like substances) havechemical properties that differ from those of mammals and othertetrapods. It is possible that they also serve quite differentfunctions in fishes than in mammals.     

Inhibition of renin secretion in the isolated rat kidney by angiotensin I.

The effect of angiotensin I on renal perfusion pressure, and on basal and isoprenaline stimulated renin secretion, was examined in the isolated perfused rat kidney. The increase in prefusion pressure associated with intrarenal infusion of angiotensin I suggested conversion of the peptide to angiotensin II within the kidney. Basal renin secretion and the stimulatory response to isoprenaline were significantly suppressed by angiotensin I. The converting enzyme inhibitor SQ 20,881, infused at 1,600 X dose of angiotensin I, partially reversed the vasoconstrictor effect of angiotensin I without altering the degree of suppression of renin secretion.     

Immunocytochemical localization of prorenin, renin, and cathepsins B, H, and L in juxtaglomerular cells of rat kidney

To examine the correlation of localization of prorenin, renin, and cathepsins B, H, and L, immunocytochemistry was applied to rat renal tissue, using a sequence-specific anti-body (anti-prorenin) that recognizes the COOH terminus of the rat renin prosegment. In serial semi-thin sections, immunodeposits for prorenin, renin, and cathepsins B, H, and L were localized in the same juxtaglomerular (JG) cells. Immunodeposits for renin were detected throughout the cytoplasm of the cells, whereas those for prorenin were detected in the perinuclear region. Immunoreactivity for cathepsin B was stronger than that for cathepsins H and L. By electron microscopy, prorenin was localized in small (immature) granules but not in large mature granules, whereas renin was localized mainly in mature granules. In serial thin sections, prorenin, renin, and cathepsin B were colocalized in the same immature granules containing heterogeneously dense material (intermediate granules). By double immunostaining, co-localization of renin with cathepsins B, H, or L was demonstrated in mature granules. The results suggest the possibility that processing of prorenin to renin occurs in immature granules of rat JG cells, and cathepsin B detected in JG cells may be a major candidate for the maturation of renin.     

Multiple defects in the renin-angiotensin system in alloxan-diabetic kidney

A defect in the renin-angiotensin system has been shown in diabetic patients and experimental animals, in particular with nephropathy or autonomic neuropathy. The mechanism for this low plasma renin activity (PRA) is poorly understood. In order to clarify this defect, the renin-angiotensin system was studied in alloxan-induced diabetic and age-match control mice. In diabetic animals, kidney renin activity (KRA) was significantly lower than that of the controls, while plasma renin substrate (PRS) concentration was slightly higher and PRA was normal. The amount of injected radiolabeled renin extracted by the kidney was normal, but the amount extracted by the liver was significantly decreased in diabetic animals. On the other hand, the degradation of the extracted renin by both the kidney and the liver was elevated as compared to the controls. This high degradation rate was accompanied by a slight increase in lysosomal protease activity in the kidneys. In in vivo studies, isoproterenol-induced PRA was 20-fold in control animals. In diabetics, isoproterenol-induced PRA was attenuated and rose only four- to fivefold over basal level. The angiotensin converting enzyme (ACE) activity in the kidney was significantly decreased in the diabetic state. It is concluded that there were multiple defects in the renin-angiotensin system in this diabetic model, namely, a depletion of renin storage with subsequent loss of maximal responsiveness to the adrenergic agonist in renin release, an elevation of intrarenal renin degradation together with a deficiency in ACE which would possibly lead to a decrease in intrarenal formation of angiotensin II.     

Maintenance of normal blood pressure and renal functions are independent effects of angiotensin-converting enzyme

Angiotensin-converting enzyme (ACE) is expressed in many tissues, including vasculature and renal proximal tubules, and its genetic ablation in mice causes abnormal renal structure and functions, hypotension, and male sterility. To test the hypothesis that specific physiological functions of ACE are mediated by its expression in specific tissues, we generated different mouse strains, each expressing ACE in only one tissue. Here, we report the properties of two such strains of mice that express ACE either in vascular endothelial cells or in renal proximal tubules. Because of the natural cleavage secretion process, both groups also have ACE in the serum. Both groups were as healthy as wild-type mice, having normal kidney structure and fluid homeostasis, though males remained sterile, because they lack ACE expression in sperm. Despite equivalent serum ACE and angiotensin II levels and renal functions, only the group that expressed ACE in vascular endothelial cells had normal blood pressure. Expression of ACE, either in renal proximal tubules or in vasculature, is sufficient for maintaining normal kidney functions. However, for maintaining blood pressure, ACE must be expressed in vascular endothelial cells. These results also demonstrate that ACE-mediated blood pressure maintenance can be dissociated from its role in maintaining renal structure and functions.     

Earliest renin containing cell differentiation during ontogenesis in the rat

Summary The differentiation of renin containing cells was studied by immunocytochemistry in normal rat fetuses by the use of highly specific renin, angiotensin I and II antisera.Renin synthesizing cells were detectable as early as the 15th day of gestation outside the nephrogen territories within the walls of mesonephrotic-gonadic and renal arteries. Intrarenal differentiation began at the 17th day and progressed along the intrarenal arterial tree. AII immunostaining appeared concomitantly in the renin containing cells and developed considerably during ontogenesis, suggesting intracellular biosynthesis.It can be suggested that in the fetus newly synthesized AH may contribute to the early systemic and renal blood pressure regulation.     

Earliest renin containing cell differentiation during ontogenesis in the rat. An immunocytochemical study

The differentiation of renin containing cells was studied by immunocytochemistry in normal rat fetuses by the use of highly specific renin, angiotensin I and II antisera. Renin synthesizing cells were detectable as early as the 15th day of gestation outside the nephrogen territories within the walls of mesonephrotic-gonadic and renal arteries. Intrarenal differentiation began at the 17th day and progressed along the intrarenal arterial tree. AII immunostaining appeared concomitantly in the renin containing cells and developed considerably during ontogenesis, suggesting intracellular biosynthesis. It can be suggested that in the fetus newly synthesized AII may contribute to the early systemic and renal blood pressure regulation.     

Contribution of angiotensin to the control of medullary hemodynamics

The unique architecture and organization of medullary vasculature permit regional regulation of medullary hemodynamics by vasoactive hormones and are conducive to the operation of the countercurrent multiplication system. Recent studies suggest that an increase in inner medullary blood flow causes medullary solute washout, which in turn decreases passive sodium transport in the thin ascending limb of Henle's loop. In canine models of chronic sodium retention accompanied by activation of the renin-angiotensin system, glomerular filtration rate (GFR), renal blood flow (RBF), and intracortical blood flow distribution were similar to those in normal dogs; however, papillary plasma flow (PPF) was markedly reduced and papillary tissue solute content was increased significantly both during hydropenia and after saline loading. During euvolemic diuresis with loop diuretics, there was an increased renin release associated with a marked reduction in PPF, despite an increase in total RBF. Direct intrarenal infusion of angiotensin II (AngII) (at a dose not affecting GFR and RBF) induced ipsilateral sodium retention, conservation of urinary concentration, and papillary ischemia. These studies provide evidence for regional regulation of medullary hemodynamics by AngII, possibly contributing to sodium retention in chronic salt-retaining states.     

Evolving concepts of the intrarenal renin-angiotensin system in health and disease: contributions of molecular biology.

Previous physiological and biochemical studies suggest the existence of an endogenous renin-angiotensin system (RAS) in the kidney. However, these data cannot exclude the contribution of the circulating RAS. Proof of the local synthesis of RAS components in the kidney has been obtained recently through the use of molecular biological techniques. Using Northern blot analysis, we have demonstrated the intrarenal expression of renin, angiotensinogen, and angiotensin-converting enzyme messenger RNAs. Employing in situ hybridization histochemistry, we have localized the intrarenal tissue sites of renin and angiotensinogen messenger RNA synthesis. Renin gene expression was found in cells of the juxtaglomerular apparatus. Angiotensinogen mRNA was primarily produced in the proximal convoluted tubule with lesser amounts in glomerular tufts and vasculature. These findings led us to hypothesize that the proximal tubule is a major site of renal Ang II synthesis and that locally synthesized Ang II might directly modulate tubular function. Both genes are subject to feedback regulation. Our studies showed that Ang II exerted a stimulatory effect on angiotensinogen but a negative feedback effect on renin gene expression. Dietary NaCl restriction stimulated the expression of both genes, although the onset of renin gene activation required more prolonged sodium chloride restriction. Furthermore, our data indicated that the sodium cation, irrespective of the anion, was primarily important in regulating renal angiotensinogen mRNA levels. Our studies also showed altered intrarenal renin or angiotensinogen expressions in pathophysiological states, e.g. in experimental heart failure and the spontaneously hypertensive rat. Taken together, these data support the existence of a intrarenal RAS and suggest its potential roles in the regulation of renal function in health and disease.